Necrotizing enterocolitis (NEC), urinary tract infections (UTIs), and sepsis are conditions that occur in very low birth weight infants (<1500g) in the first weeks of life. All three conditions are due in part to unusual microbial colonization. The initial clinical symptoms of NEC, UTIs, and sepsis are non-specific and they overlap. Diagnosis of each condition therefore often relies upon close observation paired with clinical acumen. Once diagnosis is suspected, laboratory confirmation can be time-consuming, and opportunities for treating specific conditions are wasted. Prior to confirmation of disease, infant under suspicion frequently receive broad-spectrum prophylactic antibiotic treatment that can have unintended negative consequences. A non-invasive test that assesses risk early and differentiates between NEC, UTIs, and sepsis will improve outcomes and may reduce unnecessary antibiotic exposure worldwide. To achieve this long- term goal, we aim to develop an understanding of the biochemical characteristics-biomarkers-that differ early in life between newborns that will be healthy and those that will be diagnosed with each condition. Because NEC, UTIs, and sepsis all occur at the intersection between dysbiosis and human metabolism, we focus upon urinary metabolic products as indicators of homeostasis. We have developed a protocol for extracting urine from waste diapers, identifying metabolites using high-resolution mass spectrometry, and applying statistical tools to correlate metabolites with disease states. Because we are able to obtain the necessary biomolecular information from a waste product, we are poised to study a large population of preterm newborns at a single center. This study design eliminates variables often present in multi-center studies, including microbial exposure and contamination, and makes it possible to accelerate sample acquisition. We will collect and analyze 1200 diaper samples in year one of this project. To our knowledge, no previous large-scale single-center urine-based study of preterm babies exists because of difficulty obtaining consent to collect urine from this population. We will use liquid chromatography high-resolution mass spectrometry to measure the biological molecules in urine from asymptomatic infants and infants who become ill with the study conditions, and statistical methods to draw meaningful correlations. As we uncover differences in the composition of urine, we will validate these differences as biomarkers. These biomarkers will enable earlier risk assessment and differentiation for NEC, UTIs, and sepsis in preterm infants worldwide by providing a starting point for diagnostic development. In the process of collecting these data, we will also learn about how gestational age and weight at birth, gender, food, pharmaceuticals, and maternal medications during birth impact neonatal metabolism post-partum.